This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. UVM/VGN DNA Microarray Facility: Overview The microarray core facility continues to support research for investigators both by providing services to individual research laboratories for the university and it's statewide network (see Utilization and also Publications) as well as by playing a central role in bringing emerging technologies or approaches that are important for research investigators (Scientific and Technical Changes). General background on the Microarray Facility is also included (General Background Information). Utilization March 2008 [unreadable]Feb., 2009 -Experimental design consultations with 18 investigators -Completion of 29 microarray projects (339 GeneChips) -Performed 163 RNA assessments: 79 PicoChips, 81 NanoChips, 3 small RNA's chip (new service, implemented, August, 2008) *It should be noted that a test chip was required for each target preparation before 2005 Outcomes: VCII=Vermont Immunbiology and Infectious Disease COBRE Lung=Vermont Ling Center COBRE Neuro=Vermont Neuroscience Center COBRE INBRE=Vermont Colleges Outreach=Projects completed at outreach sites Investigator Project Area Organism Chip# Project Status Affiliation Sholler Phase II Drug Trial-Nifurtimox Human 81 Ongoing Neuro Matrajt Toxoplasmosis Human 20 Manuscript in prep. VCII Budd Immunobiology 12 VCII Coe Beech tree disease Tree 2 ongoing INBRE Investigator Project Area Organism Chip# Project Status Affiliation Carlson Sports training Human 30 Analysis in process INBRE Teuscher Autoimmunity Mouse 6 Peer reviewed pub VCII Teuscher FACS Mouse 12 Bosenberg Melanoma Mouse 18 RT-qPCR validation in process Castleton Oxidative stress Yeast 6 Academic Course Outreach Kait-GMC DMSO Yeast 8 Honors thesis-complete Outreach Johnson DMSO Yeast 5 Academic Course Outreach Marlboro Photosensitivity Arabadopsis 6 Academic Course Outreach Spees Stem Cells Human 2 ongoing Neuro Teuscher Autoimmunity Mouse 6 VCII Zhao Mammary Tumorogenesis Mouse 20 Peer reviewed pub Bosenberg Melanoma Mouse 3 Validation complete Hovey Mammary Biology Porcine 36 validated by RT-qPCR Wesley, U Neuroblastoma Human 4 Validated by RT-qPCR Neuro Rand Methyl Mercury Drosophila 6 Peer reviewed pub Neuro Mossman Mesothelioma Mouse 24 Validation by RT-qPCR Lung Teuscher Autoimmune Mouse 8 VCII Carr Thyroid Cancer Human 12 Validation in progress McFadden Milk production Bovine 4 Peer reviewed pub Jaworski Embryonic Matrix Mouse 24 Manuscript in prep Neuro GMC Ox Stress Yeast 6 Academic Course Outreach Norwich TFM (lampracide) Yeast 7 Academic Course Outreach Langlais Frog virus III Xenopis 2 Preliminary INBRE Rand Methyl Mercury Drosophila 12 Neuro Langevin Tissue Mechano-transduction Mouse 4 Ongoing Neuro Microarray Generated Publications: 1: Sabo-Attwood T, Ramos-Nino M, Bond J, Butnor KJ, Heintz N, Gruber AD, Steele C, Taatjes DJ, Vacek P, Mossman BT. Gene expression profiles reveal increased mClca3 (Gob5) expression and mucin production in a murine model of asbestos-induced fibrogenesis. Am J Pathol. 2005 Nov;167(5):1243-56. PMID: 16251409 [PubMed - indexed for MEDLINE] 2: Pulver-Kaste RA, Barlow CA, Bond J, Watson A, Penar PL, Tranmer B, Lounsbury KM. Ca2+ source-dependent transcription of CRE-containing genes in vascular smooth muscle. Am J Physiol Heart Circ Physiol. 2006 Jul;291(1):H97-105. Epub 2006 Feb 3. PMID: 16461377 [PubMed - indexed for MEDLINE] 3. Muthusamy, V, Duraisamy, S, Bradbury, M, Hobbs, C, Curley, DP, Nelson, B, and Bosenberg, M. Epigenetic Silencing of Novel Tumor Supressors in Malignant Melanoma, Cancer Research;66: (23), 11187-11193, 2006 4. Chaudhry, M., Bystander effect: biological endpoints and microarray analysis. Mutat Res. 2006 May 11;597(1-2):98-112. Epub 2006 Jan 18. Review. PMID: 16414093 [PubMed - indexed for MEDLINE] 5: Zheng J, Watson AD, Kerr DE. Genome-wide expression analysis of lipopolysaccharide-induced mastitis in a mouse model. Infect Immun. 2006 Mar;74(3):1907-15. PMID: 16495566 [PubMed - indexed for MEDLINE] 6: Rolerson, E., A. Swick, L. Newlon, C. Palmer, Y. Pan, B. Keeshan, and G. Spatafora. 2006. The SloR/Dlg metalloregulator modulates Streptococcus mutans virulence gene expression. J. Bacteriol. 188:5033-5044. 7: Braas KM, Schutz KC, Bond JP, Vizzard MA, Girard BM, May V. Microarray analyses of pituitary adenylate cyclase activating polypeptide (PACAP)-regulated gene targets in sympathetic neurons. Peptides. 2007 Sep;28(9):1856-70. Epub 2007 Apr 19. PMID: 17512639 [PubMed - indexed for MEDLINE] 8: Rose P, Bond J, Tighe S, Toth MJ, Wellman TL, Briso de Montiano EM, Lewinter MM, Lounsbury KM. Genes overexpressed in cerebral arteries following salt-induced hypertensive disease are regulated by angiotensin II, JunB, and CREB. Am J Physiol Heart Circ Physiol. 2008 Feb;294(2):H1075-85. Epub 2007 Dec 21. PMID: 18156195 [PubMed - indexed for MEDLINE] 9: Finucane KA, McFadden TB, Bond JP, Kennelly JJ, Zhao FQ. Onset of lactation in the bovine mammary gland: gene expression profiling indicates a strong inhibition of gene expression in cell proliferation. Funct Integr Genomics. 2008 Aug;8(3):251-64. Epub 2008 Feb 8. PMID: 18259788 [PubMed - indexed for MEDLINE] 10: Alcorn JF, Guala AS, van der Velden J, McElhinney B, Irvin CG, Davis RJ, Janssen-Heininger YM. Jun N-terminal kinase 1 regulates epithelial-to-mesenchymal transition induced by TGF-beta1. J Cell Sci. 2008 Apr 1;121(Pt 7):1036-45. Epub 2008 Mar 11. PMID: 18334556 [PubMed - indexed for MEDLINE] 11: Rand MD, Bland CE, Bond J. Methylmercury activates enhancer-of-split and bearded complex genes independent of the notch receptor. Toxicol Sci. 2008 Jul;104(1):163-76. Epub 2008 Mar 25. PMID: 18367466 [PubMed - indexed for MEDLINE] 12: Casey T, Bond J, Tighe S, Hunter T, Lintault L, Patel O, Eneman J, Crocker A, White J, Tessitore J, Stanley M, Harlow S, Weaver D, Muss H, Plaut K. Molecular signatures suggest a major role for stromal cells in development of invasive breast cancer. Breast Cancer Res Treat. 2008 Mar 29. [Epub ahead of print] PMID: 18373191 [PubMed - as supplied by publisher] 13: Bove PF, Hristova M, Wesley UV, Olson N, Lounsbury KM, van der Vliet A. Inflammatory levels of nitric oxide inhibit airway epithelial cell migration by inhibition of the kinase ERK1/2 and activation of hypoxia-inducible factor-1 alpha. J Biol Chem. 2008 Jun 27;283(26):17919-28. Epub 2008 Apr 18. PMID: 18424783 [PubMed - indexed for MEDLINE] 14: Lu C, Pelech S, Zhang H, Bond J, Spach K, Noubade R, Blankenhorn EP, Teuscher C. Pertussis toxin induces angiogenesis in brain microvascular endothelial cells. J Neurosci Res. 2008 Sep;86(12):2624-40. PMID: 18500752 [PubMed - in process] 15. Shukla, A., MacPherson, M., Hillegass, J., Ramos-Nino, M., Alexeeva, V., Vacek, P., Bond, J., Pass, H., Steele, C., and Mossman, B., (2009) Alterations in gene expression in human mesothelialcells correlate with mineral pathogenicity , American J of Respiratory Cell and Molecular Biology, in press, 2008 Dec 18. [Epub ahead of print] 16. Dienz, O., Eaton, S.M., Bond, J.P., Neveu, W., Moquin, D., Noubade, R., Briso, E.M. Charland, C., Leonard, W.J., Ciliberto, G., Teuscher, C., Haynes, L., and Rincon, M. (2009) " The induction of antibody production by IL-6 is indirectly mediated by IL-21 produced by CD4 T cells". J Exp Med. 2009 Jan 16;206(1):69-78. Epub 2009 Jan 12. Grants received using UVM/VGN Microarray Facility generated data: Karen Lounsbury, HL67351-06 NIH, NHLBI Title: Calcium Signaling and Gene Expression in Vascular Smooth Muscle Cells, 2007-2012 Thomas McFadden, Grant# 2007-35206-17983 from the USDA Cooperative State Research, Education, and Extension Service, Title: Unilateral Frequent Milking: A Powerful Model for Identifying Biological Mechanisms Involved in Enhanced Milk Production Efficiency. Helene Langevin, Grant #RO1-AT001121, Agency: NIH National Center for Complementary and Alternative Medicine, Title: Connective Tissue Mechanotransduction. 8/01/08-04/30/13 General Background Information Staff: Scott Tighe, Senior Research Technician, 0.65FTE Tim Hunter, Facility Manager, 0.4 FTE Services Offered: n Integrated Approaches + Experimental design consultation n Assistance in RNA and DNA Extractions + LCM, Flow sorted, difficult tissues n RNA Quality/Quantity assessment n Gene Expression Profiling n DNA Mapping (SNP, LOH, CNV) n Expression profiling using 3', Exon, and Gene Arrays n Hypothesis testing: gene or pathway-based n Support letters for grant applications;text for publication n Discounted pricing on many consumables and reagents through Core Facility negotiations Scientific or technical changes: n Implementation of NuGEN Ovation and Pico WT system for target prep. + Requires minimal RNA input + Useful for FACS sorted, Micro-dissected, and exon application input + Faster turn around time n Ability to assess integrity of small RNA's and miRNA's n Addition of Qubit Spectrofluorometer for accurate quantification n Implementation of Exon and Gene Arrays using NuGEN and AmpTec reagent n New software for Exon analysis: X-Ray Boutique n Scott Tighe established protocol for extracting intact RNA from flow sorted cells. Collaborative protocol from roundtable S.Tighe chaired at NERLSCD meeting 2006, Decontaminations procedures, RNase reduction steps, extraction techniques, and QA/QC of RNA Current developmental projects underway by the Microarray Facility: [unreadable] Collaborative effort to develop stand alone software package for QA/QC of 3', Exon, and Gene arrays with Scott Tighe from UVM and John Burke at X-Ray Boutique [unreadable] Comprehensive evaluation of current target preparations on market for working with slightly to severely degraded RNA for 3', gene, and exon arrays. [unreadable] Methods to re-synthesize sense RNA for microarray from cDNA constructed without a T7 promoter -Evaluation of Modified to Amp-Tec reagents -Evaluation of sRNA reagents from Epicenter -Evaluation of SenseAMP from Genisphere 4D Professional Development: Staff attended and participated at the following meetings: -Scott Tighe, Annual Affymetrix Users Meeting, Memphis, TN, Feb. 2009 -Tim Hunter and Scott Tighe: Advances in Microarray Technology, Visited Dr. Herbert Auer's lab, collaboration on DNA Mapping, Barcelona, ESP, May, 2008 -Tim Hunter and Scott Tighe: Association of Biomolecular Resource Facilities, Memphis, TN, 2009 Facility Promotion: -Hosted Open House, June 15, 2008. Over 100 attendees -Provided Workshop on sample requirements and handling for a successful array experiment, June 15, 2008 -Hosted Seminar: "The Challenges and Successes of Implementing Next Generation Sequencing in a Share Resource Facility" June, 2008, Agnes Viale, Director of Genomics Core Laboratory, Memorial Sloan Kettering Cancer Center -Hosted Technology Seminar: "Micro RNA profiling using miScript. Presented by Doug Last, Qiagen. March 14, 2008 -Hosted Technology Seminar, Selective mRNA Amplification using AmpTec Priming Strategies, Presented by Dr. Guido Krupp, August 23, 2008 Facility Presentations: Invited Speaker: -Tim Hunter, (2008) National IDeA States of Biomedical Research Excellence, co-moderator of Core Technology Breakout Session, Washington DC, August, 2008 -Tim Hunter, Northeast Regional Life Sciences Core Directors, "Leveraging Regional Resources", Burlington, VT, Oct., 2008 -Tim Hunter, NCRR Clinical and Translational Informatics Networking Meeting, "Vermont Genetics Network Searchable Core Database", Scottsdale, AZ, Feb., 2009 -Scott Tighe, (2009) Association of Biomolecular Resource Facilities, "Common Practices for Routine RNA Handling: A Three Part Story", Memphis, TN, Feb., 2009 Invited Interview: -Tim Hunter, (2008): "Real-Time PCR: Staying Power", Biotechniques Vol 44, No.2: pp179-183 (Feb 2008) Meeting presentations (Poster): -Scott Tighe and Tim Hunter, Northeast Regional Life Sciences Core Directors, Oct. 2008, Burlington, VT, Vermont Genetics Network Microarray Facility __________________________________________________ Vermont Genetics Network Proteomics Facility. (INBRE Funded) The UVM/VGN Proteomics Facility is an interdisciplinary core facility in collaboration with the Vermont Genetics Network, the Department of Biology, the Department of Chemistry, and the College of Medicine at the University of Vermont (UVM). The Facility is located in the Marsh Life Science Building, room 311;the Cook Physical Science Building, room 113 A;and the Given Building, room C409. Equipment available includes one Applied Biosystems Voyager-DE Pro matrix-assisted laser desorption-time of flight mass spectrometer (MALDI-TOF-MS), one Thermo-Finnigan LCQ Deca XP ion trap mass spectrometer plus liquid chromatography (LC-MS), two Thermo-Finnigan LTQ linear quadrupole ion trap mass spectrometer plus nanoflow liquid chromatography (nanoLC-MS), one Thermo-Finnigan LTQ-Orbitrap hybrid mass spectrometer plus nanoflow liquid chromatography, and a Savant SpeedVac concentrator. The facility provides state-of-the-art of mass spectrometry for analyzing proteins and peptides for proteomics studies, data analysis from the proteomics measurements, training in proteomics methods, and experimental design. Pre-submission consultations are arranged with facility directors as well as post-data acquisition analysis meetings. Current and Upcoming Proteomic Services are listed below Current Proteomics Services: 1. Protein identification of samples following in-solution/in-gel digestion and using ESI LC-MS/MS. 2. Identification of common post-translational modifications of defined masses (e.g. phosphorylation, acetylation, methylation, simple glycosylation) on peptides using ESI LC-MS/MS. 3. Protein and peptide mass measurements using MALDI-TOF-MS. 4. Training of investigators in peptide quantification methods employing stable isotopes (SILAC, TMT, iTRAQ, AQUA). Peptides are subsequently quantified and identified using ESI LC-MS/MS. Upcoming Proteomics Services: 1. Measurements of intact proteins by ESI LC-MS (top-down) and ESI LC-MS/MS (middle-down). 2. Characterization of peptides with increasingly complex glycosylation. 3. Training of investigators in phosphopeptide enrichment methods including SCX-IMAC and phosphopeptide immunoprecipitation. 4. Offline HPLC fractionation of complex peptide mixtures for multi-dimensional LC-MS/MS analyses. Fiscal Year 2008-2009: The facility has run more than 1500 samples from UVM and other institutes in Vermont, which included quantitative profiling expressed proteins in tissues, and cells, large-scale mapping sites of protein phosphorylation and other post-translational modifications, and detecting low-abundance target proteins in biological and biomedical samples. Three peer-reviewed papers have been published in professional journals (see below). Four manuscripts have been submitted and 5 are in preparation. The Facility also has supported the submission of more than 20 grant proposals that have been submitted to the NIH, the NSF or other extramural funding agencies. Two NSF proposals were granted that involve heavy use of the proteomics facility. The user base for the proteomics facility is growing and for '08-'09 included 49 Faculty/Post Doctorate/Staff;24 graduate students and 16 undergraduates in this fiscal year. 24 seminars and poster presentations have been presented by Users and Staff that included VGN proteomic data. The proteomics facility has been set to charge samples for users since January 20, 2009. The first 14 samples from three PIs at UVM had been charged through UVM chart lines system, two additional investigators will be charged shortly for more than 50 samples. Additionally, on-line consultation sign-ups and on-line sample submission for users are working very well. All users'data and sample progress are on a good trajectory. The proteomics outreach module is currently going through a very productive beta testing here are UVM and there are 10 undergraduate students enrolled in this course. The outreach program carries begins with differentially-treated yeast cell sample preparation and carries the students through 1D and 2D gel separation, protein digestion, and data analysis following mass spectrometry. A regional collaboration in using SELDI-TOF and LTQ-orbitrap mass spectrometers between VGN proteomics facility and the Maine Institute for Human Genetics &Healths had been set up. Publications of UVM/VGN Proteomics Facility (2008 July-2009 present) 1. Previs MJ, VanBuren P, Begin KJ, Vigoreaux JO, LeWinter MM, Matthews DE. Quantification of protein phosphorylation by liquid chromatography-mass spectrometry. Anal Chem. 2008 Aug 1;80(15):5864-72. 2. Claude V. Gallant, Maja Sedic, Erin A. Chicoine, Teresa Ruiz, and Keith P. Mintz. Membrane morphology and leukotoxin secretion are associated with a novel membrane protein of Aggregatibacter actinomycetemcomitans. J Bacteriol. 2008 Sep;190(17):5972-80. 3. Miller MS, Lekkas P, Braddock JM, Farman GP, Ballif BA, Irving TC, Maughan DW, Vigoreaux JO. Aging enhances indirect flight muscle fiber performance yet decreases flight ability in Drosophila. Biophys J. 2008 Sep;95(5):2391-401.